Thin Layer Chromatography - Fluorescence


Many organic substances naturally fluoresce when exposed to excitation light of appropriate wavelength or can be made to form fluorescent compounds by appropriate derivatization. The plate, after development, and after treatment with the derivatizing reagents, is exposed to UV light and the solutes can be clearly seen as fluorescent spots on a light blue background. This method will detect all materials that naturally fluoresce and those substances that can be made to form fluorescent derivatives.

Fluorescence quenching is an alternative detection method using fluorescence and involves using a TLC plate that has been treated with a fluorescing reagent and, thus, the whole plate fluoresces when exposed to excitation radiation. The plate is used to separate the solutes of interest in the usual way and then is exposed to UV light. The plate is seen as a bright fluorescent sheet and the solutes as dark non-fluorescent spots where the solutes have quenched the plate fluorescence. Most organic compounds and in particular aromatic and heterocyclic compounds are detectable by this procedure.

There are a range of special reagents that have been developed to detect certain classes of compounds. Acid/base indicators can be used to detect carboxylic acids. For example the alkaline form of bromocresol green (0.25% w/v in ethyl alcohol) when sprayed on the plate will produce yellow spots on a green background for carboxylic acids. The reagent Ninhydrin (0.3% in butyl alcohol containing 3% of acetic acid) will produce purple spots on a white background with most amino acids and many amines. Aniline phthalate gives gray-black spots for reducing sugars and many natural products can be detected by spraying the plate with diphenylboricacid b-aminoethyl ester (1% in ethyl alcohol). This reagent will produce a variety of colors for different substances